Proximate composition of Vitex doniana and Saba comorensis fruits

The wild fruits of Vitex doniana and Saba comorensis were randomly collected from Pwani and Tanga regions. Laboratory analysis was done using the methods described by the Association of Official Analytical Chemists AOAC (1995 and 2000). The amount of protein was 7.13 ± 0.04% and 21.73 ± 0.02% in V. doniana fruits while the fat contents were 2.4 ± 0.00% and 1.9 ± 0.10% in V. doniana fruits. The amount of fats in S. comorensis fruits ranged from 0.00 to 0.01% for the fruit samples from both Pwani and Tanga regions, however the differences was not statistically significant (P > 0.05). The amounts of carbohydrates in V. doniana 23.98 ± 0.20% and in S. comorensis fruit samples (23.81 ± 0.38%) from Pwani Region were not statistically difference. The differences can be attributed to environmental and soil factors. S. comorensis fruit samples from Tanga had ash 4.20 ± 0.01% and moisture content 70.97 ± 0.04%. These values were higher than those observed for S. comorensis fruit samples from Pwani. The amount of ash is indicative of potential elements like sodium and potassium which are beneficial in human health for the development of bones. These fruits have significant amounts of carbohydrate and protein and hence healthy for consumption as part of human diet.


Sample preparation
The morphological characterization of fruit samples including weight and pulp weight of ripe fruits were carried out in the laboratory.An electrical analytical balance (Shimadzu ATY224; Japan) was used to measure the weight (g) of each fresh fruit and the mean weight was calculated and recorded.The weighed fruits were divided into two portions by cutting them into half using a stainless-steel knife.The pulps and seeds were removed by using a metal spoon.The pulp together with seeds were put in a dish and blended manually to separate the pulp and seeds.The seeds with residual pulp were dried in an oven at 60 oC for 5 h to completely separate seeds from residue pulp.The seeds were separated by peeling off the pulp residue and the edible parts were homogenized using mortar and pestle.The weight (g) of the pulp was measured by using an analytical balance and the mean weight was calculated.
The moisture and ash contents of the fresh fruits pulp were determined.The edible part of the fruits (mesocarp) was dried in shades for two weeks then taken to the oven overnight 65 °C.After drying the sample was ground to a powder (Fig. 1) and then analyzed for its crude fat and protein.

Determination of moisture content
The moisture content was determined using methods described by 14,15 .A clean dish was dried in an oven at 105 °C for 30 min and then cooled in a desiccator.The empty dish was weighed and 40 g of the sample was weighed and put into a dish (w1 g).The dish with a sample were then heated at 105 °C for 30 min and then cooled in desiccator.The sample was weighed (w2g) and the moisture content was calculated by using the following Eq. 1.
where by, Weight of the empty dish = (w g); Weight of dish + samples before oven drying = (w1g); Weight of the dish + sample after drying in oven = (w2g).

Determination of ash content
The method used was described by 15 .The crucible was placed on the furnace at 500 °C for 24 h to ensure that impurities on the surface of crucible are completely removed.The crucible was then cooled in the desiccator for about 30 min and then weighed.Five grams (5 g) of the sample was placed in the crucible (w3) and then heated in a furnace at 500 °C for 24 h.The sample was then cooled in the desiccator until it turns grey.The crucible with the sample and lid were weighed and calculated for ash content using Eq. 2.
where by, Weight of the dish + weight of dried sample = (w1 g); Weight of dish + weight of ash = (w3 g).www.nature.com/scientificreports/Determination of crude fat content Crude fat content was determined using method described by 15 .Fifty gram (50 g) of the sample was weighed (W 1 g) and transferred into the extraction thimble and placed in its siphon height.The weighed extraction flask was connected to the extractor carrying the thimble and 200 mL of petroleum ether was heated for 4 h.The solvent was allowed to vaporize at 60 °C and then condensed and allowed to fall drop-wise into the thimble to extract fats present in a sample.The flask with the extract was removed and the extracts were concentrated using vacuum rotary evaporator, cooled in desiccators and weighed (W 2 g).The percentage yield of ether extract was calculated based on Eq. 3.
where by, Weight of sample to be used(w); Weight of the dry flask (w1); Weight of the flask + fat after evaporation and cooled (w2).

Determination of crude protein
The crude protein was determined by conversion of organic nitrogen to ammonia based on Kjeldah method described by 16 .0.100 g of dried and finely ground sample was weighted into a 50 mL Kjeldahl flask.Two grams of potassium sulphate and copper sulphate mixture 1:1 was added into a flask.This was followed by adding 3 mL of concentrated Sulphuric acid slowly down the neck while rotating the flask and then heating gently until frothing subsided.After the digest became colorless, it was heated for 30 min to completion and allowed to cool.The digest was then diluted to 50 mL with distilled water and analyzed for nitrogen as ammonia nitrogen through spectrometric method with color reactions at 660 nm (indophenol -blue method).
Nitrogen standard (1 mL = 0.1 mg NH 4 + -N) and working standard (1 mL = 0.001 mg NH 4 + -N) were prepared from stock solutions.The combined reagent was prepared by dissolving 35 g of sodium potassium tartrate, 17.5 g sodium salicylate and 0.5 g sodium nitroprusside in 400 mL water.Then 40 mL of 50% sodium hydroxide was added, mixed and stored at 2 °C.Sodium hypochlorite solution was prepared and then used to oxidize the ammonium-nitrogen.10 mL of the working standard was pipetted into 50 mL volumetric flask to give a range from 0 to 0.001 mg NH 4 + -N.The blank was added to match the sample aliquots.5 mL of a sample was pipetted into 50 mL volumetric flask, 40 mL combined reagent was added to both standard and the sample and also 4 mL sodium hypochlorite reagent was added and diluted to volume.Then the mixture was left in the water bath at 40 °C for 10 mn.The absorbance was measured at 660 nm using UV-VIS spectrophotometer (SPECRO-UK 6305).
A calibration curve was prepared from the standard values of nitrogen and used to obtain mg of NH 4 + -N in the sample aliquot.The nitrogen (%) was calculated by using the following Eq.4; C = mg NH 4 + -N obtained from the graph.The crude protein (%) obtained by multiplying total nitrogen by conversion factor 6.25 (Eq.5).

Determination of carbohydrate
Total carbohydrate was calculated by subtracting the sum of percentage of protein, fat, ash and moisture.Carbohydrate is divided into two groups' crude fiber and nitrogen free extract (NFE).In this study only carbohydrate with crude fiber was determined, carbohydrate includes crude fiber was calculated by subtracting the sum of percentage of ash, fat, protein and moisture from hundred.The carbohydrate content was obtained by subtracting all values obtained from moisture, crude oil, crude protein, crude fiber and ash content from 100 17  www.nature.com/scientificreports/

The amount of crude protein content in V. doniana and S. comorensis
A range of 7.08% to 7.16% was observed for the amount of protein in V. doniana fruit samples from Pwani and 21.71% to 21.75% for the fruit samples from Tanga.The difference of crude protein contents in V. doniana fruits between the two regions based on two sample t-test was significant (t = 94.704,df = 2, P < 0.05).In this study S. comorensis fruits had an amount of crude protein in a range between 4.99 and 5.41% for the fruit samples from Pwani and between 5.85 and 6.19% for the fruit's samples from Tanga.The differences in ash contents on S. Comorensis fruits from the two regions based on two sample t-test was also significant (t = 5.1486, df = 2, P > 0.05) (Table 1).

The amount of crude fat content in S. comorensis and V. doniana
The amount of fats in S. comorensis fruits ranged from 0.00% to 0.01% for the fruit samples from both Pwani and Tanga.For Vitex doniana fruits the range was between 1.80% to 2.00% for the fruit samples from Tanga and 2.4 ± 0.00 for the fruit samples from Pwani.For both S. comorensis (t = -1.1094,df = 2) and V. doniana (t = 1.5443, df = 2) the differences between the Tanga and Pwani were not significant (P > 0.05).

The total carbohydrate content in S. comorensis and V. doniana
The amount of carbohydrate in S. comorensis fruit samples ranged from 23.43 to 24.19% for the fruit samples from Pwani region and 18.60-19.04%for the fruit samples from Tanga region with no significant difference between samples from the two regions based on two sample t-test (t = -0.7859,df = 2, P > 0.05) Table 1.Carbohydrate contents in V. doniana fruit samples ranged between 23.68% and 24.18% for the fruit samples from Pwani region and between 8.32% and 9.12% for the fruit samples from Tanga region.Again, the difference of carbohydrate content between two regions was not significant (t = 0.1112, df = 2, P > 0.05) (Table 2).

Variation of moisture content in the fruits of V. doniana and S. comorensis
The moisture content in S. comorensis fruits is similar to the moisture content in other wild fruits reported by 18 in Baccaurea ramiflora (70.21%).Bamigboye et al. 17 pointed out that the lowest moisture content signifies the highest dry matter content in fruits, therefore high moisture content in S. comorensis reveals lowest dry matter content.However, moisture content in Vitex doniana was close to that in Polyalthia suberosa (64.76 ± 3.91%) reported by 19 .In this study values were higher compared to those in Vitex doniana, Vitex kiniensis and Vitex fischerii reported by 20 that had the value of 39.42 ± 0.72%, 40.56 ± 0.77% and 37.74 ± 0.76%, respectively, this may be due to differences in climatic condition.The lowest moisture content in V. doniana fruits can favour long shelf life because the growth of microorganisms are not favored 21 .The percentage of moisture content in V. doniana and S. comorensis was lower compared to that in Bridela tomentosa and Carissa spinarum, 78.54 ± 1.02% and 73 ± 1.37% respectively as reported by 19 .Also some of the domesticated fruits such as Mangifera indica (82.1%) reported by 22 , Passiflora edulis (83.11%) reported by 12 and Citrus sinensis (87.1%) reported by 13 have higher moisture content than wild fruits.
Determining moisture content is important in food quality analysis because moisture affects preservation and resistance to deterioration 21 .The percentage of moisture content affects the physical and chemical properties such as color and taste of fresh food material.Therefore, the amount of moisture content in V. doniana and S. comorensis fruits can help to add amount of water in a body for a healthy skin, digestion and good flow of blood in the body. of dietary energy.The amount of carbohydrate in wild fruits contribute to higher calorific value compared to the exotic fruits.Findings from this study recommend the increase consumption of S. comorensis and V. doniana which may serve as valuable source of energy to human body.

Conclusion
Based on the results from this study wild fruits especially S. comorensis and V. doniana very nutritious having vital nutrients.However, there is no single fruit that can provide all adequate nutrients required by human being yet these wild fruits have many essential comprising nutrients such as carbohydrate, protein, crude fats, ash and moisture content.They are therefore, very potential in contributing to human health by complementing to some of the nutritional inadequacies of some of the exotic fruits.